Changes in the intestinal expression of drug metabolism-related genes in a piglet model of parenteral nutrition.

BACKGROUND: Parenteral nutrition (PN) may serve as a nutritional supportive therapy accompanied by oral medication, but the effect of PN on intestinal expression of drug metabolism-related genes remains unknown. METHODS: Twelve Bama piglets receiving PN for 14 days were used as in vivo model. Changes in intestinal drug metabolism-related genes were examined by proteomic analysis. Serum levels of fibroblast growth factor 19 (FGF19) were determined by ELISA, and the effect of FGF19 on the expression of drug metabolism-related genes was examined using murine ileum organoids. RESULTS: A total of 1063 differentially expressed proteins were identified in PN group. Of note, two drug transporters (Abcb1 and Abcc2) were significantly decreased in PN group, along with two glutathione-related drug-metabolizing enzymes, glutathione peroxidase (Gpx2) and glutathione S-transferase (Gsta1). Serum FGF19 levels were dramatically reduced in PN group. Treatment with recombinant FGF19 in vitro dose-dependently up-regulated the expression of Abcb1, Abcc2, Gpx2 and Gsta1 in organoids. CONCLUSION: Our data indicated that intestinal drug metabolism-related genes were significantly dysregulated under PN, and some of the changed genes were attributed to gut-derived FGF19.

Early downregulation of P-glycoprotein facilitates bacterial attachment to intestinal epithelial cells and thereby triggers barrier dysfunction in a rodent model of total parenteral nutrition.

Intestinal barrier dysfunction is a major complication of total parenteral nutrition (TPN). Our preliminary study revealed that intestinal P-glycoprotein (P-gp) was significantly downregulated under TPN treatment followed by disruption of barrier function, and thus the significance of early downregulation of P-gp needs to be addressed. Herein, we report a pivotal role of P-gp in the development of intestinal barrier dysfunction under TPN. Functional suppression of P-gp may facilitate bacterial attachment to intestinal epithelial cells (IECs) and thereby induce degradation of tight junctions to trigger barrier dysfunction. By using a rat model of TPN, we found early downregulation of P-gp function in ileum after 3-day TPN, followed by disruption of barrier function after 7-day TPN. By using Escherichia coli (E. coli) k88 and DH5α as type strains, we found significantly increased bacterial attachment to IECs in TPN group compared to sham. By using Caco-2 cells as an IEC model in vitro, we found that functional suppression of P-gp remarkably facilitated bacterial attachment to Caco-2 cells, leading to subsequent disruption of intestinal barrier function. Of note, Occludin was significantly downregulated by bacterial attachment when P-gp was functionally suppressed. Mechanistically, changes on Occludin were attributed to enhanced protein degradation instead of suppressed protein translation. Despite the half-life of Occludin protein being unchanged by DH5α treatment alone, it was decreased by about 40% when P-gp was simultaneously suppressed. Taken together, our findings revealed that early downregulation of intestinal P-gp under TPN may be a potential therapeutic target to prevent the development of barrier dysfunction.

Butyrate promotes the adaptation of intestinal smooth muscle cells through the yes-associated protein (YAP) pathway in a rat model of short bowel syndrome.

Our previous study demonstrated that the proliferation of human intestinal smooth muscle (ISM) cells was stimulated by butyrate through the yes-associated protein (YAP) pathway in vitro, suggesting a valuable approach for intestinal adaption of short bowel syndrome (SBS). This study was conducted to confirm these findings in vivo. Three-week-old Sprague-Dawley rats were randomly divided into the following groups: Sham group (bowel transection and reanastomosis), SB W group (80% small bowel resection/water ad libitum), and SB Bu group (80% small bowel resection/50 mM sodium butyrate ad libitum). Morphological changes were determined by hematoxylin and eosin staining; the proliferation rate of ISM cells was examined by Ki67 staining, and apoptosis was determined in the TUNEL assay. Changes in the expression of YAP and its downstream genes were evaluated by quantitative-polymerase chain reaction and western blotting. Fourteen days post-operation, a significant increase in ISM thickness was observed in the SB Bu group compared to the SB W group, accompanied by enhanced proliferation of ISM cells and suppression of apoptosis. Notably, YAP expression was also significantly increased in the SB Bu group, with a 6.5-fold increase in the proportion of YAP-positive ISM cells, 2.2-fold increase in YAP mRNA expression, and 3.4-fold increase in protein expression. In conclusion, our results suggest that butyrate promotes ISM adaption through YAP in vivo, which may be a potential therapeutic approach for SBS patients.

CELF1/p53 axis: a sustained antiproliferative signal leading to villus atrophy under total parenteral nutrition.

Intestinal villus atrophy is a major complication of total parenteral nutrition (TPN). Our previous study revealed that TPN-induced villus atrophy is accompanied by elevated expression of CUGBP, Elav-like family member 1 (CELF1); however, its mechanism of action has not been fully understood. Herein, we report a pivotal role of CELF1/p53 axis, which induces a sustained antiproliferative signal, leading to suppressed proliferation of intestinal epithelial cells (IECs). By using a rat model of TPN, we found synchronous upregulation of CELF1 and p53 in jejunum mucosa, accompanied by a 51% decrease in crypt cell proliferation rate. By using HCT-116 cells as an IEC model in vitro, we found that the expression of CELF1 altered dynamically in parallel to proliferation rate, suggesting a self-adaptive expression pattern in IECs in vitro. Furthermore, ectopic overexpression of CELF1 elicited a significant antiproliferative effect in HCT-116, Caco-2, and IEC-6 cells, whereas knockdown of CELF1 elicited a significant proproliferative effect. Moreover, cell-cycle assay revealed that ectopic overexpression of CELF1 induced sustained G2 arrest and G1 arrest in HCT-116 and IEC-6 cells, respectively, which could be abolished by p53 silencing. Mechanistically, polysomal profiling and nascent protein analysis revealed that regulation of p53 by CELF1 was mediated through accelerating its protein translation in polysomes. Taken together, our findings revealed a sustained suppression of IEC proliferation evoked by CELF1/p53 axis, which may be a potential therapeutic target for the treatment of TPN-induced villus atrophy.-Yan, J.-K., Zhang, T., Dai, L.-N., Gu, B.-L., Zhu, J., Yan, W.-H., Cai, W., Wang, Y. CELF1/p53 axis: a sustained antiproliferative signal leading to villus atrophy under total parenteral nutrition.

Fish oil-derived lipid emulsion induces RIP1-dependent and caspase 8-licensed necroptosis in IEC-6 cells through overproduction of reactive oxygen species.

BACKGROUND: Excessive cell death of enterocytes has been demonstrated to be partially associated with the intravenously-administrated lipid emulsions (LEs) during parenteral nutrition (PN) support. However, as a new generation of LE, the effect of fish oil-derived lipid emulsion (FOLE) on the death of enterocytes remains elusive. METHODS: Intestinal epithelial cells (IEC-6 cell line) were treated with FOLE (0.25-1%) for 24 h. Cell survival was measured by CCK-8 assay, and morphological changes were monitored by time-lapse live cell imaging. The expression of receptor-interacting protein 1/3 (RIP1/3) and caspase 8 was assessed by westernblot, and the formation of necrosome (characterized by the assembly of RIP1/3 complex along with the dissociation of caspase 8) was examined by immunoprecipitation. Additionally, the production of intracellular reactive oxygen species (ROS) was detected by using a ROS detection kit with an oxidation-sensitive probe (DCFH-DA). RESULTS: FOLE dose-dependently induced non-apoptotic, but programmed necroctic cell death (necroptosis) within 4-8 h after treatment. The assembly of RIP1/3 complex along with the dissociation of caspase 8 from RIP1 was observed in FOLE-treated cells. Moreover, FOLE-induced cell death was significantly alleviated by inhibiting RIP1, and was further aggravated by inhibiting caspase 8. In addition, prior to cell death the accumulation of intracellular ROS was significantly increased in FOLE-treated cells (increased by approximately 5-fold versus control, p < 0.001), which could be attenuated by inhibiting RIP1 (decreased by approximately 35% versus FOLE, p < 0.05). CONCLUSIONS: FOLE induces RIP1-dependent and caspase 8-licensed necroptosis through overproduction of ROS in vitro. Our findings may provide novel insights into the clinical applications of FOLE during PN support.

Butyrate stimulates the growth of human intestinal smooth muscle cells by activation of yes-associated protein.

Intestinal smooth muscle cells play a critical role in the remodeling of intestinal structure and functional adaptation after bowel resection. Recent studies have shown that supplementation of butyrate (Bu) contributes to the compensatory expansion of a muscular layer of the residual intestine in a rodent model of short-bowel syndrome (SBS). However, the underlying mechanism remains elusive. In this study, we found that the growth of human intestinal smooth muscle cells (HISMCs) was significantly stimulated by Bu via activation of Yes-Associated Protein (YAP). Incubation with 0.5 mM Bu induced a distinct proliferative effect on HISMCs, as indicated by the promotion of cell cycle progression and increased DNA replication. Notably, YAP silencing by RNA interference or its specific inhibitor significantly abolished the proliferative effect of Bu on HISMCs. Furthermore, Bu induced YAP expression and enhanced the translocation of YAP from the cytoplasm to the nucleus, which led to changes in the expression of mitogenesis genes, including TEAD1, TEAD4, CTGF, and Cyr61. These results provide evidence that Bu stimulates the growth of human intestinal muscle cells by activation of YAP, which may be a potential treatment for improving intestinal adaptation.

Supplementary choline attenuates olive oil lipid emulsion-induced enterocyte apoptosis through suppression of CELF1/AIF pathway.

Enterocyte apoptosis induced by lipid emulsions is a key cause of intestinal atrophy under total parenteral nutrition (TPN) support, and our previous work demonstrated that olive oil lipid emulsion (OOLE) could induce enterocyte apoptosis via CUGBP, Elav-like family member 1 (CELF1)/ apoptosis-inducing factor (AIF) pathway. As TPN-associated complications are partially related to choline deficiency, we aimed to address whether choline supplementation could attenuate OOLE-induced enterocyte apoptosis. Herein we present evidence that supplementary choline exhibits protective effect against OOLE-induced enterocyte apoptosis both in vivo and in vitro. In a rat model of TPN, substantial reduction in apoptotic rate along with decreased expression of CELF1 was observed when supplementary choline was added to OOLE. In cultured Caco-2 cells, supplementary choline attenuated OOLE-induced apoptosis and mitochondria dysfunction by suppressing CELF1/AIF pathway. Compared to OOLE alone, the expression of CELF1 and AIF was significantly decreased by supplementary choline, whereas the expression of Bcl-2 was evidently increased. No obvious alterations were observed in Bax expression and caspase-3 activation. Mechanistically, supplementary choline repressed the expression of CELF1 by increasing the recruitment of CELF1 mRNA to processing bodies, thus resulting in suppression of its protein translation. Taken together, our data suggest that supplementary choline exhibits effective protection against OOLE-induced enterocyte apoptosis, and thus, it has the potential to be used for the prevention and treatment of TPN-induced intestinal atrophy.

Urinary glutamine/glutamate ratio as a potential biomarker of pediatric chronic intestinal pseudo-obstruction.

Chronic intestinal pseudo-obstruction (CIPO) is a rare intestinal motility disorder with significant morbidity and mortality in pediatric patients. The diagnosis of CIPO is difficult, because it is clinically based on the symptoms and signs of bowel obstruction which are similar to the clinical manifestations of other gastrointestinal diseases like short bowel syndrome (SBS). Therefore, it is desirable to identify and establish new laboratory diagnostic markers for CIPO that are reliable and easily accessible. In our study we have identified the ratio of the urinary glutamine and glutamic acid as a promising biomarker for distinguishing suspected CIPO cases and simple SBS cases. The area under ROC curve was 0.83, at cutoff value = 7.04 with sensitivity of 65% and specificity of 92%.

P38 MAPK Pharmacological Inhibitor SB203580 Alleviates Total Parenteral Nutrition-Induced Loss of Intestinal Barrier Function but Promotes Hepatocyte Lipoapoptosis.

BACKGROUND & AIMS: Our previous studies have provided evidence that p38 mitogen-activated protein kinase (MAPK) is involved in total parenteral nutrition (TPN)-associated complications, but its exact effects and mechanisms have not been fully understood. This study aimed to evaluate the roles of p38 MAPK inhibitor SB203580 in the TPN-induced loss of intestinal barrier function and liver disease. METHODS: A rodent model of TPN was used to analyze the roles of SB203580 in TPN-associated complications.Intestinal barrier function was evaluated by transepithelial electrical resistance (TER) and paracellular permeability in Caco-2 cells. The palmitic acid (PA) was used to induce hepatic lipoapoptosis in vitro. The lipoapoptosis was detected using Caspase-3/7 and lipid staining. RESULTS: In the present study, we showed that SB203580 treatment significantly suppressed TPN-mediated intestinal permeability in rats. SB203580 treatment significantly inhibited IL-1ß-induced an increase in tight junction permeability of Caco-2 cells via repressing the p38/ATF-2 signaling. Unexpectedly, SB203580 treatment enhanced hepatic lipoapoptosis in the model of TPN. Palmitic acid (PA)-induced hepatic lipoapoptosis in human liver cells was significantly augmented by the SB203580 treatment. CONCLUSIONS: We demonstrate that the p38 MAPK inhibitor SB203508 ameliorates intestinal barrier function but promotes hepatic lipoapoptosis in model of TPN.

Olive Oil-Supplemented Lipid Emulsion Induces CELF1 Expression and Promotes Apoptosis in Caco-2 Cells.

BACKGROUND AND AIMS: Parenterally-administered lipid emulsion (LE) is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1) has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro. METHODS: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting. RESULTS: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE)-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF) was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation. CONCLUSION: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1.

Sodium butyrate attenuates soybean oil-based lipid emulsion-induced increase in intestinal permeability of lipopolysaccharide by modulation of P-glycoprotein in Caco-2 cells.

Down-regulation of intestinal P-glycoprotein (P-gp) by soybean oil-based lipid emulsion (SOLE) may cause elevated intestinal permeability of lipopolysaccharide (LPS) in patients with total parenteral nutrition, but the appropriate preventative treatment is currently limited. Recently, sodium butyrate (NaBut) has been demonstrated to regulate the expression of P-gp. Therefore, this study aimed to address whether treatment with NaBut could attenuate SOLE-induced increase in intestinal permeability of LPS by modulation of P-gp in vitro. Caco-2 cells were exposed to SOLE with or without NaBut. SOLE-induced down-regulation of P-gp was significantly attenuated by co-incubation with NaBut. Nuclear recruitment of FOXO 3a in response to NaBut was involved in P-gp regulation. Transport studies revealed that SOLE-induced increase in permeability of LPS was significantly attenuated by co-incubation with NaBut. Collectively, our results suggested that NaBut may be a potentially useful medication to prevent SOLE-induced increase in intestinal permeability of LPS.

Knockdown of Radixin Suppresses Gastric Cancer Metastasis In Vitro by Up-Regulation of E-Cadherin via NF-κB/Snail Pathway.

BACKGROUND/AIMS: Radixin has recently been shown to correlate with the metastasis of gastric cancer, but the pathogenesis is elusive. Adhesion proteins contribute to the regulation of metastasis, and thus this study sought to investigate the role of radixin in the migration, invasion and adhesion of gastric cancer cells, as well as its interaction with adhesion proteins in vitro. METHODS: Radixin stable knockdown human gastric carcinoma SGC-7901 cells were constructed. Alterations in the migration, invasion and adhesion ability were examined by matrigel-coated plate and transwell assays. The expression pattern of adhesion proteins, including E-cadherin, ß-catenin and claudin-1, was determined by quantitative real-time PCR and western blot. Possible involvement of NF-κB/snail pathway was also evaluated. RESULTS: Stable knockdown of radixin significantly suppressed migration and invasion, but enhanced adhesion in SGC-7901 cells. The expression of E-cadherin was manifestly increased in radixin knockdown cells, whereas the expression of ß-catenin and claudin-1 was unchanged. The nuclear exclusion of NF-κB followed by conspicuous reduction of snail expression was involved in the regulation of E-cadherin expression. CONCLUSIONS: Radixin knockdown suppresses the metastasis of SGC-7901 cells in vitro by up-regulation of E-cadherin. The NF-κB/snail pathway contributes to the regulation of E-cadherin in response to depletion of radixin.

Soybean Oil-Based Lipid Emulsion Increases Intestinal Permeability of Lipopolysaccharide in Caco-2 Cells by Downregulation of P-Glycoprotein via ERK-FOXO 3a Pathway.

BACKGROUND AND AIMS: Elevated intestinal permeability of lipopolysaccharide (LPS) is a major complication for patients with parenteral nutrition (PN), but the pathogenesis is poorly understood. Intestinal P-glycoprotein (P-gp) is one of the efflux transporters that contribute to restricting the permeability of lipopolysaccharide via transcellular route. P-gp expression may be regulated by PN ingredients, and thus this study sought to investigate the effect of PN on the expression of P-gp and to elucidate the underlying mechanism in vitro. METHODS: Caco-2 cells were treated with PN ingredients. Changes in P-gp expression and function were determined and the role of ERK-FOXO 3a pathway was studied. Transport studies of FITC-lipopolysaccharide (FITC-LPS) across Caco-2 cell monolayers were also performed. RESULTS: Among PN ingredients, soybean oil-based lipid emulsion (SOLE) exhibited significant inhibitory effect on P-gp expression and function. This regulation was mediated via activation of ERK pathway with subsequent nuclear exclusion of FOXO 3a. Importantly, P-gp participated in antagonizing the permeation of FITC-LPS (apical to basolateral) across Caco-2 cell monolayers. SOLE significantly increased the permeability of FITC-LPS (apical to basolateral), which was associated with impaired P-gp function. CONCLUSIONS: The expression and function of intestinal P-gp is suppressed by SOLE in vitro.

Neutralization of IL-6 and TNF-α ameliorates intestinal permeability in DSS-induced colitis.

The cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) have been implicated as important mediators of the inflammatory reaction in patients with intestinal inflammation. The present study was designed to investigate the roles of these cytokines on mucosal barrier function in a mouse model of acute colitis with using anti-cytokine strategies. Mice received 3% dextran sulfate sodium (DSS) in their drinking water for 7days showed morphological alteration of mucosa and increase of intestinal permeability. Administration of IL-6 monoclonal antibody (mAb) or TNF-α mAb significantly attenuated intestinal permeability. IL-6 mAb and TNF-α mAb treatment also effectively suppressed the expression of claudin-2 and myosin light chain kinase (MLCK). Taken together, we indicated that anti-IL-6 and anti-TNF-α therapy prevent intestinal permeability induced by intestinal inflammation.

CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

The IDO inhibitor coptisine ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in the kynurenine pathway (KP) of tryptophan catabolism, was recently established as one of the potential players involved in the pathogenesis of Alzheimer's disease (AD). Coptisine is a main pharmacological active constituent of the traditional Chinese medicinal prescription Oren-gedoku-to (OGT) which has therapeutic potential for the treatment of AD. Our recent studies have demonstrated that OGT significantly inhibited recombinant human IDO activity, which shed light on the possible mechanism of OGT's action on AD. Here, we characterized the effects of coptisine in an AD mouse model on the basis of its IDO inhibitory ability. Coptisine was found to be an efficient uncompetitive IDO inhibitor with a Ki value of 5.8 µM and an IC50 value of 6.3 µM. In AßPP/PS1 transgenic mice, oral administration of coptisine inhibited IDO in the blood and decreased the activation of microglia and astrocytes, consequently prevented neuron loss, reduced amyloid plaque formation, and ameliorated impaired cognition. Neuronal pheochromocytoma (PC12) cells induced with amyloid-ß peptide 1-42 and interferon-γ showed reduction of cell viability and enhancement of IDO activity, while coptisine treatment increased cell viability based on its reversal effect on the enhanced activity of IDO. In conclusion, our present findings provide further evidence supporting the critical links between IDO, KP, and AD, and demonstrate coptisine, a novel IDO inhibitor, as a potential new class of drugs for AD treatment.